A new generation of versatile chromogenic substrates for high-throughput analysis of biomass-degrading enzymes.
A new generation of versatile chromogenic substrates for high-throughput analysis of biomass-degrading enzymes.
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新一代的多功能发色底物，用于对生物质降解酶进行高通量分析。
DOI：
10.1186/s13068-015-0250-y
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发表时间：
2015
影响因子：
6.3
通讯作者：
Willats WGT
中科院分区：
文献类型：
--
作者：
Kračun SK;Schückel J;Westereng B;Thygesen LG;Monrad RN;Eijsink VGH;Willats WGT
降解或修饰多糖的酶广泛存在于原核生物和真核生物中，具有多种生物学作用和生物技术应用。基因组和分泌组测序的最新进展，以及相关的生物信息学工具，使大量的碳水化合物作用的酶被鉴定。然而，目前缺乏快速筛选这些酶的生化活性的方法，这是依赖酶的生物精炼工艺发展的严重瓶颈。我们开发了新一代多色显色多糖和蛋白质底物，可用于廉价、方便和高通量的多重检测。此外，我们还生产了生物质材料的基质，其中部分保持了植物细胞壁的复杂性。我们表明，这些底物可用于筛选糖基水解酶，溶解性多糖单加氧酶和蛋白酶的活性，并提供洞察生物质内的底物可用性。我们设想，我们已经开发的测定将主要用于大量推定的碳水化合物作用酶的第一级筛选，并且该测定有可能被纳入完全或半自动化的机器人酶筛选系统。本文的在线版本（doi：10.1186/s13068-015-0250-y）包含补充材料，可供授权用户使用。
Enzymes that degrade or modify polysaccharides are widespread in pro- and eukaryotes and have multiple biological roles and biotechnological applications. Recent advances in genome and secretome sequencing, together with associated bioinformatic tools, have enabled large numbers of carbohydrate-acting enzymes to be putatively identified. However, there is a paucity of methods for rapidly screening the biochemical activities of these enzymes, and this is a serious bottleneck in the development of enzyme-reliant bio-refining processes. We have developed a new generation of multi-coloured chromogenic polysaccharide and protein substrates that can be used in cheap, convenient and high-throughput multiplexed assays. In addition, we have produced substrates of biomass materials in which the complexity of plant cell walls is partially maintained. We show that these substrates can be used to screen the activities of glycosyl hydrolases, lytic polysaccharide monooxygenases and proteases and provide insight into substrate availability within biomass. We envisage that the assays we have developed will be used primarily for first-level screening of large numbers of putative carbohydrate-acting enzymes, and the assays have the potential to be incorporated into fully or semi-automated robotic enzyme screening systems. The online version of this article (doi:10.1186/s13068-015-0250-y) contains supplementary material, which is available to authorized users.
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影响因子：
7.4
作者：
DARVILL, JE;MCNEIL, M;ALBERSHEIM, P
通讯作者：
ALBERSHEIM, P
影响因子：
1.8
作者：
Gusakov AV;Kondratyeva EG;Sinitsyn AP
通讯作者：
Sinitsyn AP
影响因子：
6.3
作者：
Biswal AK;Soeno K;Gandla ML;Immerzeel P;Pattathil S;Lucenius J;Serimaa R;Hahn MG;Moritz T;Jönsson LJ;Israelsson-Nordström M;Mellerowicz EJ
通讯作者：
Mellerowicz EJ
影响因子：
5.2
作者：
Nyyssönen M;Tran HM;Karaoz U;Weihe C;Hadi MZ;Martiny JB;Martiny AC;Brodie EL
通讯作者：
Brodie EL
DOI：
10.1073/pnas.1323629111
发表时间：
2014-04-29
影响因子：
11.1
作者：
Agger, Jane W.;Isaksen, Trine;Westereng, Bjorge
通讯作者：
Westereng, Bjorge